Abstract The human genome is subject to constant attack by endogenous and environmental DNA damaging agents. If unrepaired, DNA lesions will give rise to mutations that in turn may lead to cancer formation. Fortunately, a complex network of DNA repair pathways operates to remove DNA lesions. To assess the biological significance of exposure to environmental DNA damaging agents, it is necessary to understand the details of the complex cellular response to DNA damage. Unlike the conserved LIG1 and LIG4 genes, lower eukaryotes lack a homolog of the mammalian LIG3 gene, which encodes at least three distinct polypeptides. Interestingly, the DNA ligase III-associated proteins, poly (ADP-ribose) polymerase 1 (PARP-1), XRCC1 and DNA polymerase (Pol) , each of which have been implicated in base excision repair and the repair of DNA single strand breaks, are also found only in higher eukaryotes. Using a modified yeast two hybrid assay, we have identified a series of XRCC1 mutants that are defective in specific protein-protein interactions. In Specific Aim 1, we will utilize these mutants to delineate the functional and biological consequences of protein-protein interactions between DNA ligase III/XRCC1 and other proteins involved in base excision and single strand break repair. Recent studies have increased the repertoire of DNA repair transactions in which DNA ligase III/XRCC1 participates. In Specific Aim 2, we will determine how DNA ligase III/XRCC1 is recruited to the DNA nucleotide excision repair machinery and whether this involves an interaction between XRCC1 and PCNA. In preliminary studies, we have identified an interaction between DNA ligase III/XRCC1 and hRad50/hMre11/Nbs. In Specific Aim 3, we will determine how DNA damage regulates this interaction and whether these proteins act together in an error- prone non-homologous end-joining sub pathway that repairs DNA double strand breaks. Interestingly, this error-prone pathway is up-regulated in cancer cells and may contribute to their characteristic genomic instability. In Specific Aim 4, we will identify and characterize small molecule inhibitors of DNA ligase III. We envision that that these inhibitors will not only be valuable reagents for elucidating the cellular functions of the LIG3 gene products but also may serve as lead compounds for the development of novel anti-cancer agents.